Our long term goal is to understand the control of the determinants of initiation of DNA replication in mammalian cells. The system we have developed for this purpose rests on the identification of the replication initiation sites in the mouse immunoglobulin heavy chain gene (Igh) locus. We have established that the timing of replication at this locus differs radically between cells in the early and late stages of B lineage development. In cells in the early stages of development the entire Igh locus replicates early in the S phase, while in the late stages part of the locus replicates later in S phase. We inferred that the number and location of replication initiation sites changes during B cell development. We propose now to track these changes in cells of the B lineage throughout the course of differentiation. We will also examine the relationship between histone modifications and the activation of new replication initiation sites. We will rely on the new approach that we have developed, as set out in the objectives of our previous application, for the study of DNA replication in single-copy gene loci in mammalian cells; we have termed this Single Molecule Analysis of Replicated DNA (SMARD). This approach has recently enabled us to analyze the replication initiation sites in the single copy Igh locus of mammalian cells. It further affords us now a means of following, through all the stages of B cell development, the replication of the Igh locus in both cell lines and primary cells. The latter have not previously been amenable to study because of the large number of cells in each developmental stage demanded by the conventional, electrophoretic, method of analysis. Both normal and malignant cells will be studied. Our studies will establish whether indeed replication origin usage changes with development and ultimately will make it possible to resolve the question of whether the replication fork direction in the Igh locus governs some B cell specific processes such as VDJ recombination, isotype switching and somatic hypermutation. We will use SMARD to localize and characterize the developmentally regulated replication initiation sites in B cell lines and primary cells representing successive stages of differentiation. We will study DNA replication throughout the Igh locus in order to identify replication initiation sites that are active in pro and pre B cells and silenced in cells representing later stages of B cell development. We will study the acetylation status of histones at activated and silenced developmentally regulated initiation sites in the Igh locus in B cell lines and primary cells. Finally, we will use gene targeting in ES cells to determine the impact of transcription, possibly through the modification of chromatin structure, on the activation of replication origins in primary B lineage lineage cells.